Candidate Genes Research Articles

Unravelling the role of key genes involved coffee leaf rust resistance

The biotrophic fungus Hemileia vastatrix is the pathogen responsible for coffee leaf rust, a devastating disease in several coffee-producing countries. Despite the importance of studying the interaction between Coffea and H. vastatrix, a more comprehensive understanding of the mechanisms involved in this pathosystem is necessary. The role of eight candidate genes was analyzed aiming at identifying and validating new important coffee resistance genes and understanding their interaction with H. vastatrix. These genes were identified in the most important sources of coffee resistance, the Híbrido de Timor CIFC 832/2 and CIFC 832/1. Previous works found six resistance genes and, in our research, other two new genes were identified in BAC clones and validated by RT-qPCR during compatible and incompatible interactions between Coffea and H. vastatrix. An interactome approach was performed using Coffea-H. vastatrix and Coffea-Coffea proteins to better understand the biological process and the interaction of the host-pathogen. Two networks of interactions from the compiled data were built focused on candidate genes associated with pre-haustorial resistance (12 and 24h.a.i) in coffee plants against the pathogen. The results showed, for the first time, differentially expressed proteins (DEPs) positively regulated in the incompatible interaction Coffea-H. vastatrix. These coffee proteins interact with each other and with secreted and/or transmembrane pathogen proteins. The obtained results also show that DEPs found are involved in important plant defense pathways such as pathways associated with the response to wounds, signaling, regulation of the innate immune response and the transmembrane receptor protein serine/threonine kinase pathway. The present work shows the involvement of genes in both pathogen recognition and signaling cascades, which act in pre-haustorial defense mechanisms of HdT coffee. Therefore, the candidate genes analyzed, together with the biological processes elucidated, have the potential to contribute to the development of new control strategies against the fungus H. vastatrix within coffee breeding programs aiming to develop cultivars with durable resistance.

Human-specific insights into candidate genes and boosted discoveries of novel loci illuminate roles of neuroglia in reading disorders.

Reading disorders (RD) are human-specific neuropsychological conditions associated with decoding printed words and/or reading comprehension. So far only a handful of candidate genes segregated in families and 42 loci from genome-wide association study (GWAS) have been identified that jointly provided little clues of pathophysiology. Leveraging human-specific genomic information, we critically assessed the RD candidates for the first time and found substantial human-specific features within. The GWAS candidates (i.e., population signals) were distinct from the familial counterparts and were more likely pleiotropic in neuropsychiatric traits and to harbor human-specific regulatory elements (HSREs). Candidate genes associated with human cortical morphology indeed showed human-specific expression in adult brain cortices, particularly in neuroglia likely regulated by HSREs. Expression levels of candidate genes across human brain developmental stages showed a clear pattern of uplifted expression in early brain development crucial to RD development. Following the new insights and loci pleiotropic in cognitive traits, we identified four novel genes from the GWAS sub-significant associations (i.e., FOXO3, MAPT, KMT2E and HTT) and the Semaphorin gene family with functional priors (i.e., SEMA3A, SEMA3E and SEMA5B). These novel genes were related to neuronal plasticity and disorders, mostly conserved the pattern of uplifted expression in early brain development and had evident expression in cortical neuroglial cells. Our findings jointly illuminated the association of RD with neuroglia regulation-an emerging hotspot in studying neurodevelopmental disorders, and highlighted the need of improving RD phenotyping to avoid jeopardizing future genetic studies of RD.

Therapeutic Potential of CRISPR/Cas in Hashimoto's Thyroiditis: A Comprehensive Review.

Hashimoto's thyroiditis (HT) is a commonly occurring illness of autoimmune endocrine origin. It is usually present in the pediatric age group along with other well-known diseases, such as type 1 insulin-dependent diabetes. The defining feature of this disease is the immune-- mediated attack on the thyroid gland resulting in the destruction of thyroid tissues and cells. Given that HT frequently affects family members, it is well-recognized that individuals are genetically predisposed to this disease. Patients with HT also display a significantly increased risk for several different cancers, justifying the eminent need for the development of therapies for managing and treating HT. Gene editing has made several advancements in the field of molecular biology and has turned out to become a promising approach to correct several autoimmune diseases. Currently, CRISPR/Cas, a nuclease-based editing technique, is publicized as a promising tool for curing several genetic diseases and cancers. However, very limited research has been conducted as of now on autoimmune disease management and cure via CRISPR/Cas technique. This review provides an account of the potential candidate genes associated with Hashimoto's thyroiditis, and only a few animal and human models have been generated via the CRISPR/Cas gene editing technique. Mouse models of autoimmune thyroiditis generated through the CRISPR/Cas gene editing technique by targeting the candidate genes will provide us with a deeper insight into the pathophysiology of HT and further pave the way for the immunomodulation of HT via gene editing.

Overexpression of E3 ligase RING finger protein BrRING509 enhances salt stress tolerance in transgenic Arabidopsis plants

Salinity is an important abiotic stress that has an impact on crop yields and quality. Ubiquitin-mediated protein degradation enables plants to effectively withstand environmental stresses. The E3 ligases have been identified as a crucial component of the ubiquitination pathway, which regulates plant responses to abiotic stresses. The RING finger E3 ligase family of proteins has a critical role in plant growth and response to many abiotic stresses. In this study, we identified an important salt-responsive gene, BrRING509 in Brassica rapa. It is localized throughout the plasma membrane and nucleus. The functional results suggest that BrRING509 significantly increases salt stress tolerance in Arabidopsis (Arabidopsis thaliana) transgenic plants. Overexpression of BrRING509 could increase the survival and root length of transgenic seedlings under different salt concentrations. Additionally, ectopic expression of BrRING509 significantly increases the expression level of salt-responsive genes, including AtRD22 and AtRD29A in Arabidopsis transgenic plants subjected to salt stress treatments. The findings of this study indicate that BrRING509 plays a positive role in salt stress tolerance and might be an important candidate gene for the adaptation of Brassica plants in salt-stress environments.

Mapping and candidate gene analysis of QTLs for grain shape in a rice chromosome segment substitution line Z485 and breeding of SSSLs.

The online version contains supplementary material available at 10.1007/s11032-024-01480-x.

A bZIP type transcription factor from Picrorhiza kurrooa modulates the expression of terpenoid biosynthesis genes in Nicotiana benthamiana

bZIP family of transcription factors are DNA-binding proteins which bind ACGT motifs in the upstream promoter region of genes. bZIP proteins play significant roles in regulation of plant growth, secondary metabolism, development, defense and response to various abiotic and biotic stresses. Up to now, there is no report on molecular and functional characterization of bZIP transcription factors in Picrorhiza kurrooa, a medicinally important endangered herb of north western Himalayan region. The species is widely used in several commercially available drug formulations like livomap, livocare and livplus for treatment of liver disorders. The medicinal properties of P. kurrooa are due to bioactive picrosides, synthesized by isoprenoid pathway. The promoters of regulatory genes viz., Pkdxs and Pkhmgr of picroside biosynthesis pathway have been reported to contain ACGT cis-acting elements (bzip motifs) at different locations. In this study, the functionality of ACGT motifs was confirmed by electrophoretic mobility shift assay (EMSA). Full length PkbZIP gene was cloned through RACE method. In silico analysis revealed PkbZIP contained a conserved signature bZIP domain (N-x7-R/K-x9-L-x6-L-x6-L). Transcriptional activation and subcellular localization assay confirmed nuclear localization of PkbZIP. EMSA with purified PkbZIP confirmed in vitro binding of PkbZIP to ACGT elements in Pkdxs and Pkhmgr promoters. The expression pattern of PkbZIP positively correlated with the picroside (active medicinal component of the plant) content in different tissues, at 15 °C and under light conditions. Transient overexpression in Nicotiana benthamiana suggested a role of PkbZIP in terpenoid biosynthesis. PkbZIP might prove as potential candidate gene to be used in genetic engineering program for enhancing the picroside content in P. kurrooa.

Transcriptome analysis indicates the mechanisms of BmNPV resistance in Bombyx mori midgut

Bombyx mori nucleopolyhedrovirus (BmNPV) caused serious economic losses in sericulture. Analyzing the molecular mechanism of silkworms (B. mori) resistance to BmNPV is of great significance for the prevention and control of silkworm virus diseases and the biological control of agricultural lepidopteran pests. In order to clarify the defense mechanisms of silkworms against BmNPV, we constructed a near isogenic line BC8 with high resistance to BmNPV through the highly BmNPV-resistant strain NB and the highly BmNPV-susceptible strain 306. In this study, RNA-Seq technique was used to analyze the transcriptome level differences in the midgut of BC8 and 306 following BmNPV infection. A total of 1350 DEGs were identified. Clustering analysis showed that these genes could be divided into 8 clusters with different expression patterns. Functional annotations based on GO and KEGG analysis indicated that they were involved in various metabolism pathways. Finally, 32 BmNPV defense responsive genes were screened. They were involved in metabolism, reactive oxygen species (ROS), signal transduction and immune response, and insect hormones. The further verification shows that HSP70 should participate in resistance responses of anti-BmNPV. These findings have paved way in further functional characterization of candidate genes and subsequently can be used in breeding of BmNPV resistance dominant silkworms.

Spinach genomes reveal migration history and candidate genes for important crop traits.

Spinach (Spinacia oleracea) is an important leafy crop possessing notable economic value and health benefits. Current genomic resources include reference genomes and genome-wide association studies. However, the worldwide genetic relationships and the migration history of the crop remained uncertain, and genome-wide association studies have produced extensive gene lists related to agronomic traits. Here, we re-analysed the sequenced genomes of 305 cultivated and wild spinach accessions to unveil the phylogeny and history of cultivated spinach and to explore genetic variation in relation to phenotypes. In contrast to previous studies, we employed machine learning methods (based on Extreme Gradient Boosting, XGBoost) to detect variants that are collectively associated with agronomic traits. Variant-based cluster analyses revealed three primary spinach groups in the Middle East, Asiaand Europe/US. Combining admixture analysis and allele-sharing statistics, migration routes of spinach from the Middle East to Europe and Asia are presented. Using XGBoost machine learning models we predict genomic variants influencing bolting time, flowering time, petiole color, and leaf surface texture and propose candidate genes for each trait. This study enhances our understanding of the history and phylogeny of domesticated spinach and provides valuable information on candidate genes for future genetic improvement of the crop.

Non-Pathogenic Pythium Induced the Resistance in Hydroponic Lettuce Against Root Rot Disease

Hy droponics is a common soilless type of culture. It is clean and gives high-yield crop production. Lettuce is the most widely grown leafy vegetable in hydroponics. The Pythium species causes root rot, a serious disease in lettuce. In this study, we investigated an effective elicitor to promote resistance in lettuce after pathogen infection. To evaluate the plant defense gene response profile, the expression stability of five candidate genes was studied using RT-qPCR. The LsPR1 and LsLTC2 gene expression were higher in lettuce leaves treated with non-pathogenic Pythium PyASR23 than in roots compared to untreated plants. To assess the disease, the rate of relative gene expression of LsLTC2, LsPR-1b like, LsERF, LsAOS, and LsDEF was associated with Pythium root rot pathogenesis. As a result, it was revealed that alterations in these gene expressions were a defense response to Pythium infection, which increased after pre-treatment with various elicitors for 24 hours. When the chemically stimulated lettuce was infected with pathogenic PySR31, the expression of these genes increased dramatically. These results suggested that non-pathogenic PyASR23 might be employed as an elicitor in lettuce roots and contribute to disease control by activating plant defense gene expression against the Pythium pathogen.

Identification of candidate genes for Fusarium head blight resistance from QTLs using RIL population in wheat.

Fusarium head blight (FHB) stands out as one of the most devastating wheat diseases and leads to significantly grain yield losses and quality reductions in epidemic years. Exploring quantitative trait loci (QTL) for FHB resistance is a critical step for developing new FHB-resistant varieties. We previously constructed a genetic map of unigenes (UG-Map) according to the physical positions using a set of recombinant-inbred lines (RILs) derived from the cross of 'TN18 × LM6' (TL-RILs). Here, the number of diseased spikelets (NDS) and relative disease index (RDI) for FHB resistance were investigated under four environments using TL-RILs, which were distributed across 13 chromosomes. A number of 36 candidate genes for NDS and RDI from of 19 stable QTLs were identified. The average number of candidate genes per QTL was 1.89, with 14 (73.7%), two (10.5%), and three (15.8%) QTLs including one, two, and 3-10 candidate genes, respectively. Among the 24 candidate genes annotated in the reference genome RefSeq v1.1, the homologous genes of seven candidate genes, including TraesCS4B02G227300 for QNds/Rdi-4BL-4553, TraesCS5B02G303200, TraesCS5B02G303300, TraesCS5B02G303700, TraesCS5B02G303800 and TraesCS5B02G304000 for QNds/Rdi-5BL-9509, and TraesCS7A02G568400 for QNds/Rdi-7AL-14499, were previously reported to be related to FHB resistance in wheat, barely or Brachypodium distachyon. These genes should be closely associated with FHB resistance in wheat. In addition, the homologous genes of five genes, including TraesCS1A02G037600LC for QNds-1AS-2225, TraesCS1D02G017800 and TraesCS1D02G017900 for QNds-1DS-527, TraesCS1D02G018000 for QRdi-1DS-575, and TraesCS4B02G227400 for QNds/Rdi-4BL-4553, were involved in plant defense responses against pathogens. These genes should be likely associated with FHB resistance in wheat.

Salicylic acid modulates secondary metabolism and enhanced colchicine accumulation in long yellow daylily (Hemerocallis citrina Baroni)

Abstract Salicylic acid (SA) is an essential phytoregulator that is widely used to promote the synthesis of high-value nutraceuticals in plants. However, its application in daylily, an ornamental plant highly valued in traditional Chinese medicine, has not been reported. Herein, we investigated the exogenous SA-induced physiological, transcriptional, and biochemical changes in long yellow daylily (LYD). We found that 2 mg/L foliar SA treatment significantly improved LYD plant growth and yield. Transcriptome sequencing and differentially expressed genes analysis revealed that the phenylpropanoid biosynthesis, isoquinoline alkaloid biosynthesis, sulfur metabolism, plant hormone signal transduction, and tyrosine metabolism were significantly induced in SA-treated leaves. Many transcription factors and antioxidant system-related DEGs were induced under the SA treatment. Biochemical analyses showed that the leaf contents of soluble sugar (SS), soluble protein (Cpr), ascorbic acid (AsA), and colchicine were significantly increased by 15.15% (from 30.16±1.301 to 34.73±0.861 mg/g), 19.54% (from 60.3±2.227 to 72.08±1.617 mg/g), 30.45% (from 190.1±4.56 to 247.98±11.652 μg/g), and 73.05% (from 3.08±0.157 to 5.33±0.462 μg/g), respectively, under the SA treatment. Furthermore, we identified 15 potential candidate genes for enhancing the growth, production, and phytochemical content of LYD. Our results provide support for the bioaccumulation of colchicine in yellow daylily and valuable resources for biotechnological-assisted production of this important nutraceutical in Hemerocallis spp.

Identification and functional marker development of SbPLSH1 conferring purple leaf sheath in sorghum.

We identified a SbPLSH1gene conferring purple leaf sheath in sorghum (sorghumbicolor(L.) Moench)and developed a functional markerfor it. The purple leaf sheath of sorghum, a trait mostly related to anthocyanin deposition, is a visually distinguishable morphological marker widely used to evaluate the purity of crop hybrids. We aimed to dissect the genetic mechanism for leaf sheath color to mine the genes regulating this trait. In this study, two F2 populations were constructed by crossing a purple leaf sheath inbred line (Gaoliangzhe) with two green leaf sheath inbred lines (BTx623 and Silimei). Based on the results of bulked-segregant analysis sequencing, bulk-segregant RNA sequencing, and map-based cloning, SbPLSH1 (Sobic.006G175700), which encodes a bHLH transcription factor on chromosome 6, was identified as the candidate gene for purple leaf sheath in sorghum. Genetic analysis demonstrated that overexpression of SbPLSH1 in Arabidopsis resulted in anthocyanin deposition and purple petiole, while two single-nucleotide polymorphism (SNP) variants on the exon 6 resulted in loss of function. Further haplotype analysis revealed that there were two missense mutations and one cis-acting element mutation in SbPLSH1, which are closely associated with leaf sheath color in sorghum. Based on the variations, a functional marker (LSC4-2) for marker-assisted selection was developed, which has a broad-spectrum capability of distinguishing leaf sheath color in natural variants. In summary, this study lays a foundation for analyzing the genetic mechanism for sorghum leaf sheath color.

ABCD4 is associated with mammary gland development in mammals.

Mammary gland development is a critical process in mammals, crucial for their reproductive success and offspring nourishment. However, the functional roles of key candidate genes associated with teat number, including ABCD4, VRTN, PROX2, and DLST, in this developmental process remain elusive. To address this gap in knowledge, we conducted an in-depth investigation into the dynamic expression patterns, functional implications, and regulatory networks of these candidate genes during mouse mammary gland development. In this study, the spatial and temporal patterns of key genes were characterized in mammary gland development. Using time-series single-cell data, we uncovered differences in the expression of A bcd4, Vrtn, Prox2, and Dlst in cell population of the mammary gland during embryonic and adult stages, while Vrtn was not detected in any cells. We found that only overexpression and knockdown of Abcd4 could inhibit proliferation and promote apoptosis of HC11 mammary epithelial cells, whereas Prox2 and Dlst had no significant effect on these cells. Using RNA-seq and qPCR, further analysis revealed that Abcd4 can induce widespread changes in the expression levels of genes involved in mammary gland development, such as Igfbp3, Ccl5, Tlr2, and Prlr, which were primarily associated with the MAPK, JAK-STAT, and PI3K-AKT pathways by functional enrichment. These findings revealed ABCD4 as a candidate gene pivotal for regulating mammary gland development and lactation during pregnancy by influencing PRLR expression.

KIN10-mediated HB16 protein phosphorylation and self-association improve cassava disease resistance by transcriptional activation of lignin biosynthesis genes.

Cassava bacterial blight significantly affects cassava yield worldwide, while major cassava cultivars are susceptible to this disease. Therefore, it is crucial to identify cassava disease resistance gene networks and defence molecules for the genetic improvement of cassava cultivars. In this study, we found that MeHB16 transcription factor as a differentially expressed gene in cassava cultivars with contrasting disease resistance, positively modulated disease resistance by modulating defence molecule lignin accumulation. Further investigation showed that MeHB16 physically interacted with itself via the leucine-Zippe domain (L-Zip), which was necessary for the transcriptional activation of downstream lignin biosynthesis genes. In addition, protein kinase MeKIN10 directly interacted with MeHB16 to promote its phosphorylation at Ser6, which in turn enhanced MeHB16 self-association and downstream lignin biosynthesis. In summary, this study revealed the molecular network of MeKIN10-mediated MeHB16 protein phosphorylation improved cassava bacterial blight resistance by fine-tuning lignin biosynthesis and provides candidate genes and the defence molecule for improving cassava disease resistance.

Genetic Mapping for Leaf Shape and Leaf Size in Non-Heading Chinese Cabbage by a RIL Population

Leaves are the predominant photosynthetic and edible organs in non-heading Chinese cabbage (Brassica campestris ssp. chinensis, NHCC), contributing significantly to yield, appearance, and desirability to consumers. However, the genetic basis of leaf shape and size in non-heading Chinese cabbage remains unclear. In this study, we developed a RIL population using ‘Maertou’, with slender leaves and narrow petioles, and ‘Suzhouqing’, with oval leaves and wide petioles, to construct a genetic linkage map and detect QTLs. To obtain stable and reliable QTLs, the 11 leaf-related traits, including the leaf length, leaf width, and fresh weight of the lamina and petiole and the thickness of petiole was observed on two locations—while the leaf shape, petiole shape, index of lamina/petiole length, and index of petiole fresh weight were calculated based on 7 leaf-related traits. QTL mapping illustrated that a total of 27 QTLs for leaf-related traits were preliminarily detected. The candidate genes were annotated and several genes involved in leaf development and leaf shape appeared in the overlapping regions of multiple loci, such as KRP2, GRF4, ARGOS, and SAUR9. This study lays the foundation for further exploration of the genetic mechanisms and development of effective molecular markers for leaf shape and size in NHCC.

Genome-wide screening of meta-QTL and candidate genes controlling yield and yield-related traits in barley (Hordeum vulgare L.).

Increasing yield is an important goal of barley breeding. In this study, 54 papers published from 2001-2022 on QTL mapping for yield and yield-related traits in barley were collected, which contained 1080 QTLs mapped to the barley high-density consensus map for QTL meta-analysis. These initial QTLs were integrated into 85 meta-QTLs (MQTL) with a mean confidence interval (CI) of 2.76 cM, which was 7.86-fold narrower than the CI of the initial QTL. Among these 85 MQTLs, 68 MQTLs were validated in GWAS studies, and 25 breeder's MQTLs were screened from them. Seventeen barley orthologs of yield-related genes in rice and maize were identified within the hcMQTL region based on comparative genomics strategy and were presumed to be reliable candidates for controlling yield-related traits. The results of this study provide useful information for molecular marker-assisted breeding and candidate gene mining of yield-related traits in barley.

Genome-wide association analysis uncovers rice blast resistance alleles of Ptr and Pia

A critical step to maximize the usefulness of genome-wide association studies (GWAS) in plant breeding is the identification and validation of candidate genes underlying genetic associations. This is of particular importance in disease resistance breeding where allelic variants of resistance genes often confer resistance to distinct populations, or races, of a pathogen. Here, we perform a genome-wide association analysis of rice blast resistance in 500 genetically diverse rice accessions. To facilitate candidate gene identification, we produce de-novo genome assemblies of ten rice accessions with various rice blast resistance associations. These genome assemblies facilitate the identification and functional validation of novel alleles of the rice blast resistance genes Ptr and Pia. We uncover an allelic series for the unusual Ptr rice blast resistance gene, and additional alleles of the Pia resistance genes RGA4 and RGA5. By linking these associations to three thousand rice genomes we provide a useful tool to inform future rice blast breeding efforts. Our work shows that GWAS in combination with whole-genome sequencing is a powerful tool for gene cloning and to facilitate selection of specific resistance alleles for plant breeding.

Application and prospect of BSA method based on bibliometrics in crop breeding.

In order to understand the progress and frontier in the application of BSA(bulked segregant analysis) method in crop breeding and to reflect objectively the contribution of different countries, institutions and researchers in this field at home and abroad, this study analyzed 2111 items in the WOS (Web of Science) database from 2000 to 2023 and 446 items in the CNKI (China National through Knowledge Infrastructure) database from 2003 to 2023, regarding the researches of the application of BSA in crop breeding, basing on bibliometric analysis methods using CiteSpace software including keyword co-occurrence analysis, highlight word analysis, keyword clustering analysis, clustering timeline analysis and author co-citation. The results showed that there was an consistent increasing trend in the publication number of the application of BSA in crop breeding both in the domestic and foreign journals year by year. Ranking of the top countries according to the number of publications was China, the United States and India. The Huazhong Agricultural University displayed the highest number of publications in the CNKI database, while the Chinese Academy of Agricultural Sciences was found to have the highest number of publications in the WOS database. The published articles related to the application of BSA in crop breeding abroad mainly focused on the disciplines such as plant science, agronomy, horticulture and genetics, while those in China mainly concentrated on such disciplines as plant science, plant protection, horticulture and biology. The top three authors in terms of influence in the field of appling BSA in crop breeding were Michelmore RW, Kosambi DD and Li H, while Michelmore RW, Lander ES and Li H had closer cooperations with other authors. The top three crops relating to the studies of BSA were rice(Oryza sativa), soybean(Glycine max), corn(Zea mays L.) with the hot spot traits of disease resistance and plant height domestically. The top three crops involving the studies of BSA were rice, Arabidopsis thaliana and wheat(Triticum aestivum L.) with hot spot traits of disease resistance abroad. Up to now, BSA was mainly used to localize and functionally verify the candidate genes linking target traits and the mutated genes in crops in the domestical documents, while the foreign published studies based on BSA were mainly focused on the fine mapping and functional verification of target trait genes aiming at the revelation of genetic mechanisms in crops. Research frontier analysis indicated that rice, peanuts(Arachis hypogaea L.), upland cotton(Gossypium hirsutum L.) would be the main objects of studies concerning application of BSA in crop breeding with the hot topics of crop mutants and crop metabolites in the future.

Analysis of copy number variants detected by sequencing in spontaneous abortion.

The incidence of spontaneous abortion (SA), which affects approximately 15-20% of pregnancies, is the most common complication of early pregnancy. Pathogenic copy number variations (CNVs) are recognized as potential genetic causes of SA. However, CNVs of variants of uncertain significance (VOUS) have been identified in products of conceptions (POCs), and their correlation with SA remains uncertain. Of 189 spontaneous abortion cases, trisomy 16 was the most common numerical chromosome abnormality, followed by monosomy X. CNVs most often occurred on chromosomes 4 and 8. Gene Ontology and signaling pathway analysis revealed significant enrichment of genes related to nervous system development, transmembrane transport, cell adhesion, and structural components of chromatin. Furthermore, genes within the VOUS CNVs were screened by integrating human placental expression profiles, PhyloP scores, and Residual Variance Intolerance Score (RVIS) percentiles to identify potential candidate genes associated with spontaneous abortion. Fourteen potential candidate genes (LZTR1, TSHZ1, AMIGO2, H1-4, H2BC4, H2AC7, H3C8, H4C3, H3C6, PHKG2, PRR14, RNF40, SRCAP, ZNF629) were identified. Variations in LZTR1, TSHZ1, and H4C3 may contribute to embryonic lethality. CNV sequencing (CNV-seq) analysis is an effective technique for detecting chromosomal abnormalities in POCs and identifying potential candidate genes for SA.

Lack of evidence for GWAS signals of exfoliation glaucoma working via monogenic loss-of-function mutation in the nearest gene.

Exfoliation syndrome (XFS) is a systemic disease of elastin-rich tissues involving a deposition of fibrillar exfoliative material (XFM) in the anterior chamber of the eye, which can promote glaucoma. The purpose of this study was to create mice with CRISPR/Cas9-induced variations in candidate genes identified from human genome-wide association studies (GWAS) and screen them for indices of XFS. Variants predicted to be deleterious were sought in the Agpat1, Cacna1a, Loxl1, Pomp, Rbms3, Sema6a, and Tlcd5 genes of C57BL/6J mice using CRISPR/Cas9-based gene editing. Strains were phenotyped by slit-lamp, SD-OCT imaging, and fundus exams at 1-5 mos of age. Smaller cohorts of 12-mos-old mice were also studied. Deleterious variants were identified in six targets; Pomp was recalcitrant to targeting. Multiple alleles of some targets were isolated, yielding 12 strains. Across all genotypes and ages, 277 mice were assessed by 902 slit-lamp exams, 928 SD-OCT exams, and 358 fundus exams. Homozygosity for Agpat1 or Cacna1a mutations led to early lethality; homozygosity for Loxl1 mutations led to pelvic organ prolapse, preventing aging. Loxl1 homozygotes exhibited a conjunctival phenotype of potential relevance to XFS. Multiple other genotype-specific phenotypes were variously identified. XFM was not observed in any mice. This study did not detect XFM in any of the strains. This may have been due to species-specific differences, background dependence, or insufficient aging. Alternatively, it is possible that the current candidates, selected based on proximity to GWAS signals, are not effectors acting via monogenic loss-of-function mechanisms.